The need to develop new vaccines against mucosal pathogens has compelled a search for adjuvants that can effectively stimulate mucosal immunity to vaccine proteins, most of which are poor immunogens. Toll- like receptors (TLRs) and gangliosides are considered "adjuvant receptors" because their interaction with certain classes of noxious microbial molecules generates potent immunomodulatory signals in antigen- presenting cells (APCs) that mobilize adaptive immunity. An ideal adjuvant should thus stimulate adjuvant receptors without, however, inducing toxic effects. This proposal will investigate the adjuvant activities of a novel ganglioside-binding TLR2 agonist, recently identified by our group. The molecule under investigation is the pentameric B subunit of a unique E. coli enterotoxin, designated Type Mb(LT-llb) and quite different from Type I enterotoxins such as cholera toxin. In contrast to the intact toxin, the B subunit (designated LT- llb-B) induces TLR2-dependent activation of transcription factor NF-KB, which plays a central role in the induction of immunoregulatory cytokines and costimulatory molecules on APCs, such as dendritic cells. The overall hypothesis is that LT-llb-B exhibits immunomodulatory activity, which requires the cooperation of GD1a ganglioside and TLR2, promotes antigen-presenting cell function, and induces mucosal immunity. We thus aim to elucidate the immunomodulatory mechanisms of LT-llb-B and demonstrate its mucosal adjuvanticity. Specifically, using immunological, biophysical, and cell imaging techniques, we will determine cooperative interactions between gangliosides and TLR2 in response to LT-llb-B, and the importance of these interactions in upregulating dendritic cell-mediated functional costimulation to CD4+T cells. Moreover, it will be determined if these adjuvant mechanisms of LT-llb-B correlate with induction of mucosal adjuvant activity in vivo. The proposed studies are supported by a plethora of preliminary findings and are facilitated by the availability of (a) engineered wild-type and single point substitution mutants of LT-II molecules, (b) model cell systems where expression of receptors of interest is manipulated and (c) a well-established mouse mucosal immunization model. Using these models and state-of-the-art interdisciplinary techniques, the generated data will form the basis for our ultimate objective which is to establish LT-llb-B as a safe and effective adjuvant in vaccine formulations against oral or other mucosal pathogens.